A series of heme proteins have been discovered in the saliva of the blood-sucking insect Rhodnius prolixus that act as vasodilators and anti-platelet aggregation and anti-histaminic agents when injected into the victim. These heme proteins are novel Fe(III) proteins that are reversible carriers of NO and tight binders of histamine. During the first four-year period of this grant, significant progress has been made in characterizing the most abundant nitrophorin, NP1. This protein has been found to be unique in terms of structure (a beta-barrel with a heme in its mouth), reduction potential and resistance to autoreduction in the presence of excess NO. For the second period, the goals of the project are to characterize all four nitrophorins, NP1-NP4, and a number of selected site-directed mutants. Specific aims include the investigation of the electrochemistry of NP2 and NP3, for comparison to NP1 and NP4, in the absence and presence of NO, and of selected site-directed mutants of these four proteins. These investigations are aimed at determining the effect of the carboxylate and other protein side chains in the heme pocket on the reduction potentials of the nitrophorins and their NO complexes. The NMR spectra of both the high-spin and low-spin complexes of NP2 will be compared to the spectra of the same complexes of NP1. The 3D structure of NP2 will be determined by NMR spectroscopy in order to investigate the dynamics of this beta barrel protein in each of these forms and to measure the pKas of the carboxylates near the heme. Detailed EPR investigations of NP1-NP4 in the presence of NO and of imidazole, cyanide and histamine complexes of NP1-NP4 will be performed. The Mossbauer spectra of NP1-NO and NP2-NO in the Fe(III)NO and FE(II)NO forms will be obtained. FTIR studies of NP2-NO in the Fe(III)NO and Fe(II)NO forms as well as detailed resonance Raman studies in the absence and presence of NO will be performed. These will be used to characterize heme distortion modes. MCD spectroscopic studies of NP1 and NP1-NO and other nitrophorins will be continued in order to assign the optical transitions of these proteins. The spectroscopic and electrochemical properties of the nitrophorin from Cimex lectularius will be characterized if this gene can be expressed. The finding that insects from two families contain heme proteins that carry NO reversibly yet have very different protein sequences and molecular weights suggests that the scenario of storage of NO bound to heme may be a relatively common scenario among blood-sucking insects. The insights gained form these studies should allow us to provide perspective on the biochemical, physiological and pharmacological roles that NO plays in human health.